Cells are continually exposed to factors, such as intracellular reactive species and environmental agents that are capable of causing DNA damage. Single bases in DNA can be chemically damaged by a variety of mechanisms, the most common ones being deamination, oxidation, and alkylation. These modifications can affect the ability of the base to hydrogen-bond, resulting in incorrect base-pairing, and, as a consequence, mutations in the DNA. For example, incorporation of adenine across from 8-oxoguanine (right) during DNA replication causes a G:C base pair to be mutated to T:A.
Agents that damage DNA include ionizing radiation such as gamma rays and X-rays, ultraviolet rays, especially the UV-C rays (˜260 nm) that are absorbed strongly by DNA, but also the longer-wavelength UV-B that penetrates the ozone shield; high-reactive oxygen radicals produced during normal cellular respiration as well as by other biochemical pathways; chemicals in the environment, including some found in cigarette smoke; some plant and microbial products, e.g., the aflatoxins produced in moldy peanuts; and chemicals used in chemotherapy, especially chemotherapy of cancers.
The potentially mutagenic consequences of DNA damage are minimized by DNA repair pathways, which are broadly characterized into three forms: base excision repair (BER), mismatch repair (MMR), and nucleotide excision repair (NER). Deficiencies in DNA damage repair underlie the pathogenesis of cancer as well as many genetic disorders, such as Xeroderma pigmentosum, Cockayne syndrome, and Ataxia-telangiectasia.
BER is a cellular mechanism that repairs damaged DNA throughout the cell cycle. BER is important for removing damaged bases that could otherwise cause mutations by mispairing or lead to breaks in DNA during replication. BER is initiated by DNA glycosylases, which recognize and remove specific damaged or inappropriate bases. These are then cleaved by an endonuclease. The resulting single-strand break can then be processed by either short-patch (where a single nucleotide is replaced) or long-patch BER (where 2-10 new nucleotides are synthesized).
Steps and agents involved in BER include:                1. Removal of the damaged base (estimated to occur some 20,000 times a day in each cell) by a DNA glycosylase. There are at least 8 genes encoding different DNA glycosylases with each enzyme responsible for identifying and removing a specific kind of base damage.        2. Removal of its deoxyribose phosphate in the backbone, producing a gap. There are two genes encoding enzymes with this function.        3. Replacement with the correct nucleotide. This relies on DNA polymerase β.        4. Ligation of the break in the strand. Two enzymes are known that can do this; both require ATP to provide the needed energy.        
Defects in a variety of DNA repair pathways lead to cancer predisposition. For example, deletion mutations in BER genes have been shown to result in a higher mutation rate in a variety of organisms, implying that loss of BER could contribute to the development of cancer. Indeed, somatic mutations in Pol β have been found in 30% of human cancers, and some of these mutations lead to transformation when expressed in mouse cells. Mutations in the DNA glycosylase MYH are also known to increase susceptibility to colon cancer.
To date, most of the cancer therapies that target DNA repair pathways are substances that inhibit DNA repair in cancer cells in order to enhance the effects of DNA-damaging chemotherapies and radiotherapies. Fewer attempts have been made to improve or accelerate DNA repair in order to reduce the consequences of DNA damage after it has occurred in order to prevent or treat disease, although compositions comprising T4 endonuclease V have been examined as a potential therapy for skin cancer (Cafardi and Elmets. Expert Opin. Biol. Ther., 8(6): 829-38 (2008)).
BER is an essential biological process for cells in combating a variety of DNA base lesions, which are the most common form of DNA damage in human cells. However, no simple, fast, and direct cellular-level high throughput measurement of BER capacity exists as of yet.
Current methods of BER measurement are either inaccurate (e.g. monitoring decreases in DNA damage) or too limited in their application (e.g. plasmid transfection-based measurement).
While plasmid transfection-based measurement of DNA repair in cells is a recent development, certain limitations prevent this method from being used at a high throughput scale. First, it involves the complex processes of tissue culture and plasmid transfection into single cultured mammalian cells. Furthermore, this technology is restricted by the limited availability of plasmids containing various types of DNA lesions and the inefficiency of introducing the plasmids into different types of mammalian cells. The cell transfection-based assay on DNA repair also prevents its application in measuring DNA repair capacity in human tissue.
Thus, there is a need for compositions and methods to measure DNA repair in cells and in animals, as well as for assays to quickly and accurately measure the capacity DNA base lesion repair in cells and animals.